Protection of the sanitary nature of drinking water is a concern worldwide. While numerous sources of microbial contamination exist, a principle concern is the introduction of pathogenic intestinal protozoa such as Cryptosporidium and Giardia into the water supply. These microorganisms are particularly troublesome, as they are often present in the form of cysts and oocysts. The process of encystment renders the organisms environmentally resistant. Such intestinal pathogens are often introduced into the environment by the direct deposit of human or animal feces (both domestic and feral), or through purposeful or accidental discharge of sewage or wastewater into the lake, stream, or aquifer which supplies raw water to be treated for the purpose of rendering it potable. In addition to testing the drinking water for the presence of microorganisms, tests upstream can often identify the source of contamination. In addition to drinking water tests, tests of water where swimming or bathing is expected is also often necessary to prevent spread of disease, as is illustrated by the closure of much of the Western shore of Michigan's Lake Saint Clair during the summer of 1993 due to fecal contamination from sewage overflow and concurrent weather conditions which maintained rather than ameliorated the contamination in windward areas.
The current proposed test for Cryptosporidium and Giardia is set forth in ASTM D-19 Proposal P229, "Proposed Test Method for Giardia Cysts and Cryptosporidium Oocysts in Low-Turbidity Water by a Fluorescent Antibody Procedure." The proposed test method of the Environmental Protection Agency (EPA), United States, was published in 59 FED. REGISTER No. 28, 6416-6427 (Feb. 10, 1994), and is substantially similar to the ASTM P229 method. Both methods are herein incorporated by reference.
In the ASTM P229 method, water to be tested is directed through a cartridge filter containing a spiral wound 25.4 cm (10 inch) long depth filter. The recommended water volume is 380 L (100 gallons), and is directed through the filter cartridge, the nominal 1 .mu.m spiral wound filter element retaining suspended matter (but see below) and passing the water in which the sediment is suspended. When the necessary quantity of water has traversed the filter, the cartridge is disassembled, and the water present in the cartridge housing and the cartridge itself are transferred to a plastic sample bag, e.g., a Zip-Lock.RTM. bag, and preferably double bagged, for transportation to the laboratory.
In the lab, the filter cartridge is cut apart with a knife or scalpel and the yarn wrapping and sediment separated into three portions, an interior, relatively sediment-free portion, an intermediate portion, and an outside, heavily sedimented portion. The yarn in each of these portions is washed successively with the same three 1.0 L volumes of eluting solution, the most interior yarn being washed first. The washing step consists of massaging the yarn in the eluting solutions by hand, or alternatively adding the yarn to a stomaching bag (alternative EPA procedure) and homogenizing in a stomacher followed by hand massaging and homogenizing a second time.
The eluate, containing sediment and any microorganisms trapped by the filter, is centrifuged for 10 minutes at 1050.times. g in 250 ml centrifuge bottles in a swinging bucket centrifuge, the supernatant discarded to waste and the solids combined, resuspended, and n recentrifuged to produce a pellet whose volume is measured, a portion of which is resuspended and tested for the presence of Cryptosporidium and Giardia by standard molecular methods, in this case fluorescent antibody procedures. Additional information relative to these molecular methods as well as other methods which may, in general, be useful is disclosed by Abdallah M. Isa, "Elisa Technology", ENCYCLOPEDIA OF MICROBIOLOGY, Vol. 2, pp. 59-62 (1992); J. Lederberg, Ed., "Indirect Fluorescent Antibody Tests and Other Immunomicroscopic Methods", ENCYCLOPEDIA OF MICROBIOLOGY, Vol. 2, pp. 163-164 (1992); E. Baron, L. Peterson and S. Finegold, DIAGNOSTIC MICROBIOLOGY, Bailey & Scott's; pp. 1015-1085; D. Jones, "Polymerase Chain Reaction (PCR)", ENCYCLOPEDIA OP MICROBIOLOGY, Vol. 3, pp. 443-449 (1992); and J. Conroy, R. Stevens and K. Hechemy, "Enzyme Immunoassay", ENCYCLOPEDIA OF MICROBIOLOGY, Vol. 3, pp. 87-92 (1992) which are incorporated by reference.
The prior art methods of testing leave much to be desired. Among the deficiencies are the following:
Filter cartridges and housings from different manufacturers are not fully inter-changeable; PA1 Filter cartridges may be installed incorrectly; PA1 Filter housings must be cleaned thoroughly between uses, which is both time-consuming as well as offering the potential for cross-contamination; PA1 Filter "pore size," although nominally 1 .mu.m, spans a wide range, allowing considerable sediment and microorganisms, if present, to pass through; PA1 Housing water and filter cartridge are stored in plastic bags which are susceptible to leakage through damage or improper closure, presenting both cross-contamination, transportation, and loss of sample risks; PA1 Installation and removal must be performed with protective equipment (latex gloves). PA1 The filter element must be manually cut-apart with a knife or scalpel, which poses a risk of infection to the technician, even when wearing latex gloves; PA1 The washing procedure is laborious and uses a large quantity of eluate; PA1 The washing procedure is inefficient, resulting in relatively low recovery of microorganisms, and in addition is highly variable; PA1 The centrifugation of eluate requires large centrifuge bottles which are preferably discarded after use to avoid cross-contamination; and PA1 The combination of centrifuged samples for further concentration runs the risk of sample loss and/or contamination.
The foregoing deficiencies are associated with the basic nature of the filter cartridge and housing and its use in the field. However, upon arrival at the laboratory, further serious deficiencies arise. For example:
The foregoing are but some of the deficiencies associated with the prior art process. As an indication of how much of an impact these deficiencies may have on microorganism quantitation, in a round robin test in which water containing a known quantity of challenge microorganisms was "quantitated" using the proposed ASTM procedure, an average of less than 3% (EPA Contract No. 68-C3-0365, WA No. 2-2, p. 21) of cryptosporidium microorganisms were recovered.
It would be desirable to provide a method of isolation of microorganisms from liquids which minimizes the potential for contamination and cross-contamination; which substantially prevents leakage during shipment; which maximizes sediment and microorganism retention; which is more economical of time and capital; which substantially decreases risk of infection of laboratory personnel; which allows quantitation with smaller sample size; which may provide smaller quantities of eluate with which to work; and which provides the opportunity for greater accuracy and reliability of the quantitation.